1. Field Of The Invention
The invention relates to a method for detecting nitric oxide and nitrosonium equivalents in a biological sample, involving subjecting a biological fluid to photolysis prior to chemiluminescence detection by reaction with ozone. The invention further relates to a method for detecting S-nitrosothiols and S-nitroso-proteins in a biological sample, which involves treating the samples with mercury and a protein-precipitating agent, respectively, prior to chemiluminescence detection, and comparing with the chemiluminescence signal generated by a sample absent said treatment.
2. Brief Description Of The Background Art
Endothelium-derived relaxing factor (EDRF), is a product of the normal endothelial cell, and has both vasodilatory and antiplatelet properties (Furchgott, R. F. et al., Nature 288:373-376 (1980); Moncada, S. et al., Biochem. Pharmacol. 38:1709-1713 (1989); Azuma, H. et al., Brit. J. Pharmacol. 88:411-415 (1986) and Radomski, M. W. et al., Brit. J. Pharmacol. 92:639-642 (1987)). Pharmacologic studies suggest that disease states as varied as septic shock, hyperhomocysteinemia, atherosclerosis, and hypoxia-induced pulmonary hypertension may be associated with abnormal concentrations of EDRF in the vascular milieu (Westenberger, U. et al., Free Rad. Res. Comm. 11:167-168 (1990); Yamamoto, H. et al., J. Clin. Invest. 81:1752-1758 (1988); Dinh-Xuan, A. T. et al., Engl. J. Med. 324:1539-1547 (1991)). This bioactive substance is believed to be equivalent to nitric oxide, or a chemical congener or adduct thereof (Palmer, R. M. G. et al., Nature 327:524-525 (1987); Ignarro, L. J. et al., Proc. Natl. Acad. Sci. 84:9265-9269 (1987)). Among the species of importance as biological adducts of nitric oxide are S-nitrosothiols-, which are adducts with the sulfhydryl groups of amino acids, peptides, and proteins.
It has been demonstrated that nitric oxide and authentic EDRF react with free thiol groups of proteins under physiologic conditions in vitro, to form S-nitroso-proteins. These nitric oxide adducts have bioactivities which are comparable to nitric oxide, but exhibit half-lives on the order of hours, significantly longer than that of EDRF (Stamler, J. S. et al., Proc. Natl. Acad. Sci. 89:444-448 (1992)). The single free cysteine present on serum albumin (cysteine 32) is particularly reactive toward nitrogen oxides (most likely nitrosonium ion) under physiologic conditions, primarily because of its anomalously low pK, and its abundance in plasma, where it accounts for an approximate 0.5 mM thiol concentration.
Under normal circumstances, the concentration of nitric oxide in blood or plasma is believed to be quite low (in the 1 nM range) and its half life of the order of 0.1 second. Its high degree of reactivity toward oxygen and redox metals, in conjunction with its extremely short half-life, have made the routine measurement of blood levels in both normal and disease states most difficult by standard methods, such as chemiluminescence spectroscopy, electron paramagnetic resonance spectroscopy, or differential absorbance spectroscopy of hemoglobin (Martin, W. et al., J. Pharmacol. Exp. Therap. 237:529-538 (1986); Downes, M. J. et al., Analyst 101:742-748 (1976); Kelm, M. et al., Circ. Res. 66:1561-1575 (1990); Arroyo, C. M. et al., Free Rad. Res. Comm. 14:145-155 (1991) and Goretsky J. et al., J. Biol. Chem. 263:2316-2323 (1988)). In fact, it is generally assumed in the field that such measurements are not feasible by currently used methods.
Nitrosonium (NO.sup.+) is a short lived species which is too unstable to exist freely in biological systems, and felt to be non-detectable by chemiluminescence. Nitric oxide exists in the S-nitrosothiol adduct, not as nitric oxide but rather as a nitrosonium equivalent. Thus, it behaves chemically in a manner which more closely resembles NO.sup.+, than NO.cndot. (nitric oxide). S-nitrosothiols have not been detected by chemiluminescence and it is generally not appreciated in the art that nitrosonium is a biologically relevant species.
Chemiluminescence spectroscopy has been used, in particular, to detect nitric oxide in studies of EDRF in vitro. By the standard analysis, however, samples to be tested are subjected to extensive chemical pretreatment that precludes the discrimination of free nitric oxide, from nitrosonium, labile adducts or from higher oxidation states of nitrogen. Given the demonstration by these investigators of the importance of nitric oxide adducts (nitrosonium adducts), such as S-nitrosothiols and S-nitroso-proteins, in biological systems, a need exists for an analytical method which enables one to distinguish these compounds from nitric oxide, and thereby detect and quantify them in biological samples.